In recent years, with rapid progress of chemical biology, the involvement of genes in various diseases has been understood with a fair degree of precision, and medical cares targeted at genes have attracted attention.
With respect to DNA, currently, SNPs (which is an abbreviation of single nucleotide polymorphisms, and a general term for a difference of a single code (single base) in genes) attract attention. The reason is as follows. By classifying SNPs, it is possible to predict the prevalence rates of many diseases, and the effects or sensitivities of individuals to medical agents, and furthermore, it is possible to perform perfect identification of an individual because there absolutely exist no plural human beings having completely the same SNPs on the planet, even parent and child or brothers.
The applicant of the present invention has proposed a gene examination device and a gene examination method for separating a sample DNA utilizing a conjugate DNA, as a method for examining DNAs having different portions in base sequences such as the above-mentioned SNPs (refer to Japanese Published Patent Application No. 2002-340858).
Hereinafter, the conventional method will be described with reference to FIGS. 6 to 8. FIG. 6 is a diagram illustrating the construction of a common capillary electrophoresis device, FIG. 7 is a state diagram in a capillary tube of the capillary electrophoresis device, and FIG. 8 is a diagram illustrating the relationship between a sample DNA and a conjugate DNA in the conventional method.
With reference to FIG. 6, in a capillary electrophoresis device 100, a first container 131 in which a positive electrode 133 is disposed and a second container 132 in which a negative electrode 134 is disposed are connected by a capillary tube 130 that is filled with a conjugate DNA including a buffer solution 11. Then, as shown in FIG. 7, a sample DNA 200 is injected into the capillary tube 130 that is filled with the conjugate DNA including the buffer solution 11. The conjugate DNA 210 is, as shown in FIG. 8, obtained by combining a marker DNA 212 having a base sequence that is complementary to a portion of a base sequence of a target DNA included in the sample DNA 200 directly with a non-electrophoresis material 211 that hardly moves during electrophoresis, such as linear polymer. The sample DNA 200 is obtained by mixing a first sample DNA 201 which includes, in a portion of its base sequence, the target DNA having a base sequence that is complementary to the conjugate DNA 210, and a second sample DNA 202 having a portion of a base sequence that is not complementary to the conjugate DNA.
Thereafter, a voltage is applied to the both electrodes 133 and 134 by a variable power supply 135 to make the sample DNA 200 in the capillary tube 130 perform electrophoresis, and the sample DNA 200 is separated due to a difference in affinities to the conjugate DNA 210 between the first sample DNA 201 and the second sample DNA 202 in the sample DNA 200.
Next, a description will be given of a method for pseudo-immobilizing the conjugate DNA 210 in the capillary tube 130.
Among DNAs, there exist DNA that forms a double strand and DNA that forms a single strand. Among four bases possessed by DNA, i.e., adenine (A), thymine (T), cytosine (C), and guanine (G), A and T, or G and C are easily bonded to each other, and when DNA forms a double strand, A and T, or G and C are paired with each other. Accordingly, when one of DNAs forming a double strand has a base sequence of 5′-ATCGCGT-3′, the other DNA has a base sequence of 5′-ACGCGAT-3′.
The conjugate DNA to be used in the conventional capillary electrophoresis device utilizes the above-mentioned complementary relation of DNA in order to separate the sample DNA. That is, as shown in FIG. 8, a DNA sequence that is complementary to the target DNA to be detected in the sample DNA 200 is given to the marker DNA 212 in the conjugate DNA 210.
For example, assuming that the first sample DNA 201 as the target DNA in the sample DNA 200 is mutant DNA, and the DNA sequence of the mutant DNA includes 5′-ATCGCGT-3′ while the DNA sequence of the wild DNA as the second sample DNA included in the sample DNA 200 is 5′-ATCACGT-3′, a base of the mutant DNA 201 and a base of the wild DNA 202 differ from each other at the underlined portions. At this time, assuming that the sequence of the marker DNA 212 in the conjugate DNA 210 is 5′-ACGCGAT-3′, the wild DNA 202 becomes not complementary to the marker DNA 212 in the conjugate DNA 210 at the underlined portion. Thereby, in the sample DNA 200 bonded to the conjugate DNA 210, the entire bonding force of the mutant DNA 201 becomes larger than that of the wild DNA 202, and the mutant DNA 201 moves with a delay from the wild DNA during electrophoresis.
By the way, the above-mentioned DNA sample 200 is formed by extracting DNA from blood or the like by destroying cells, and amplifying a portion including a target DNA sequence by PCR or the like. At this time, while the number of bases of the conjugate DNA 210 is determined according to the number of bases and the base sequence pattern of the portion to be amplified, the number of bases of the target DNA to be amplified by PCR or the like is about 50, in which it is probabilistically considered that the same DNA sequences do not exist, in human genome DNA comprising about three billion of base pairs.
The conjugate DNA 210 is formed as follows. When the non-electrophoresis material 211 is linear polymer made of acrylamide, the 5′ end of the marker DNA 212 having a sequence complementary to the target DNA sequence in the sample DNA 200 is vinylated, and the marker DNA 212 is mixed at a predetermined rate into the acrylamide monomer, and further, ammonium persulfate as a polymerization starter and tetramethylethylenediamine as a polymerization agent are mixed thereto, and the resultant solution is left still for two hours.
The conjugate DNA 210 formed as described above is filled in the capillary tube 130 of the conventional capillary electrophoresis device 100, and the sample DNA 200 is injected into the conjugate DNA 210 to make the sample DNA 200 move from the second container 123 to the first container 131 by electrophoresis, whereby interaction between the conjugate DNA 210 and the sample DNA 200 occurs not only in the vicinity of the wall surface of the capillary tube 130 but also inside the capillary tube 130, and the sample DNA 200 can be separated into the wild DNA 202 and the mutant DNA 201 by a difference in movement speeds between the wild DNA 202 and the mutant DNA 201 in the sample DNA 200 that is bonded to the conjugate DNA 210 by the interaction, and consequently, gene abnormality of SNPs can be discriminated easily and accurately in short time.
As described above, the conventional conjugate DNA 210 is constituted such that the end of the marker DNA 212 having a base sequence that is complementary to the base sequence of the target DNA as a detection target in the sample DNA 200 is bonded to the non-electrophoresis material 211, conjugate DNAs as many as the target DNAs are required, and therefore, formation of a conjugate DNA must be carried out from the beginning every time the detection target changes, resulting in an immense amount of time until the result of measurement is obtained.
Further, the separation performance of the conventional conjugate DNA 210 is determined by the bonding force of the complementary hydrogen bonding with the sample DNA, the bonding force of the marker DNA 212 and the sample DNA 200 varies depending on the length or the sequence pattern of the base sequence of the marker DNA 212 in the conjugate DNA 210, and therefore, it is necessary to search for various conditions for appropriately separating the sample DNA 200, every time the base sequence of the target DNA in the sample DNA 200 changes.
To be specific, among the four bases of DNA, adenine (A) and thymine (T) are paired while cytosine (C) and guanine (G) are paired as described above, and adenine (A) and thymine (T) are bonded by two hydrogen bonds while cytosine (C) and guanine (G) are bonded by three hydrogen bonds. Accordingly, even when the length of the marker DNA 212 is constant, the bonding force of the sample DNA 200 and the marker DNA 212 varies depending on the sequence pattern of the sample DNA 200. For example, when the base sequence of the marker DNA 212 as the complementary bonding portion comprises 6 bases, there exist 12 hydrogen bonds at minimum (all A-T bonds) to 18 hydrogen bonds at maximum (all C-G bonds), and therefore, the bonding force varies very much even when the length of the base sequence of the complementary bonding portion is constant.
However, when performing separation of the sample DNA 200 by electrophoresis, the items that can be controlled after preparation of the conjugate DNA 210 on the device 100 side are only the voltage during electrophoresis and the measurement temperature, and such control on the device 100 side has a limitation in controlling the bonding force of the sample DNA 200 and the marker DNA 212 in the conjugate DNA 210. In order to solve this problem, conventionally, the conjugate DNA 210 is formed according to the base sequence of the detection target DNA included in the sample DNA 200, whereby the bonding force between the target DNA included in the sample DNA 200 and the marker DNA 212 having the sequence complementary to the target DNA is appropriated.
As a method thereof, for example, the length of the marker DNA 212 in the conjugate DNA 210, which is the complementary bonding portion to the sample DNA, is increased or reduced.
In this method, however, the bonding force between the sample DNA 200 and the marker DNA 212 cannot be minutely controlled.
In order to solve this problem, conventionally, minute control of the bonding force between the sample DNA 200 and the conjugate DNA 210 is realized by controlling, for every sample DNA 200, the amount of the bonding control agent included in the conjugate DNA 210, the viscosity of the non-electrophoresis material 211, the amount of the sample DNA 200, or the amount of the conjugate DNA 210.
The electrophoresis speed of the sample DNA 200 becomes slower as the amount of the bonding control agent is larger, the viscosity of the non-electrophoresis material 211 is higher, the amount of the conjugate DNA 210 is larger, the voltage during electrophoresis is lower, and the measurement temperature is lower, and thereby the bonding force between the sample DNA 200 and the conjugate DNA 210 in the hermetically sealed flow path is increased.
However, as described above, it requires an immense amount of labor and time to estimate various patterns in which the amount of the bonding control agent included in the conjugate DNA, the viscosity of the non-electrophoresis material 211, the amount of the sample DNA 200, and the amount of the conjugate DNA 210 are complicatedly combined for each sample. DNA, and search for an optimum electrophoresis condition by examination.